Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein
| Autor: | Jie Xu, Luo Sun, Inca Ghosh, Ming-Qun Xu |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2004 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 36, Iss 6, Pp 976-981 (2004) |
| Druh dokumentu: | article |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/04366ST02 |
| Popis: | We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase. |
| Databáze: | Directory of Open Access Journals |
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